Lecture 2 Flashcards
thinckness of band on southern blot means what?
amount of DNA present in band
what is RFLP used for?
to track a mutation in a family
allele markers (RFLP) are specific for what reason?
spouses come from different family background, if a spouse have the allele will not necessaryly have the mutation
ASO hybridization
based method to detect sequence varients (mutations of poly morphisms)
what type of prob is used in ASO?
oligonucleotides
how many probes to test for a gene?
2, one for wild tyoe one for mutatnt allele
what if theres not a hybridization in the mutatnt allele
negative result
type of target DNA used in ASO?
genomic DNA. dont need to run target DNA through a gel.
probes labeled with what?
p32
probes labeled with what?
p32
how many spots per individual in ASO?
- each with different probe
why short probes are used?
even with mismatch it will not bond. more specific results. if the probe is too long it will still bind even with mismatch
classic mutation used to talk about point mutation?
sickle cell disease
conservative point mutation
amino acid changed because of mutation will have similar properties with each other
non conservative mutation
amino acid replaced with another with different properties. ex. GLU to VAL
what types of questions on ASO hybridization?
only to analyse the dots. will not be tested on methods.
how is dna amplified in pcr
exponentially.
number of copies produced per dna molecule cycle?
2 to the power of number of cycles 2^n
Taq polymerase
heat resistant polymerase from bacteria grwon in hot spring
problem with Taq polymerase?
no proofreading
advantage of Pfu polymerase?
has proofreading capacity, but it is slow
PCR methods
start with dna polymerase, primers, dna sequence. and dNTP , magnesium
- heat to separate dna strands 95 degrees
- cool to 55-60 to allow primers to aneal
- dna polymerase from the 3 prime end of each primer at 72 degrees
optimal temp. for Taq polymerase?
72 degrees
themocyclers
metal block to insert test tubes equiped with timers and heating and cooling elements.
they automatically heat and cool solution.
denaturation, anealing, and extension temperatures for pcr?
95, 55, 72, respectively
what can pcr be used for?
RFLP
sequencing
direct mutation detection
determining repeat length
limits for diagnostic of fragment size with pcr?
1 kb fragment
how to desing pcr primers?
- have to have 2 primers, inside the region of interest. the primer is part of what is being synthesized
- place primer on 3’ end of each strand
3
general size of primer?
about 20 nt long
how sequence of one strand relate to the other strand?
same as other. because other is complementary to it
reverse primer sequence related to sequnce of other strand?
complementary.
reverse primer sequence related to sequnce of other strand?
complementary.
to use PCR to amplify RNA?
need to turn it into DNA first, resverse transcriptase to synthesize DNA from mRNA
preparation of cDNA by PCR?
get mRNA and reverse transcriptase to rapidly get bunch of dna from it. no need to making cDNA library anymore
RT-PCR?
reverse transcriptase PCR. product ismeasured as reaction is happening, not at the end.
measures the production of a fluorescent-labeled PCR produc in real time as the cycles are progressing
Real time PCR or qPCR
quantitative method.
is standard pcr quantitative? why
no, towards the end of reaction doubling is not perfect
